Interpreting second-harmonic generation images of collagen I fibrils

Biophys J. 2005 Feb;88(2):1377-86. doi: 10.1529/biophysj.104.047308. Epub 2004 Nov 8.


Fibrillar collagen, being highly noncentrosymmetric, possesses a tremendous nonlinear susceptibility. As a result, second-harmonic generation (SHG) microscopy of collagen produces extremely bright and robust signals, providing an invaluable tool for imaging tissue structure with submicron resolution. Here we discuss fundamental principles governing SHG phase matching with the tightly focusing optics used in microscopy. Their application to collagen imaging yields several biophysical features characteristic of native collagen structure: SHG radiates from the shell of a collagen fibril, rather than from its bulk. This SHG shell may correspond to the supporting element of the fibril. Physiologically relevant changes in solution ionic strength alter the ratio of forward-to-backward propagating SHG, implying a resulting change in the SHG shell thickness. Fibrillogenesis can be resolved in immature tissue by directly imaging backward-propagating SHG. Such findings are crucial to the design and development of forthcoming diagnostic and research tools.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Algorithms
  • Animals
  • Collagen Type I / analysis
  • Collagen Type I / chemistry
  • Collagen Type I / ultrastructure*
  • Fibrillar Collagens / analysis
  • Fibrillar Collagens / chemistry
  • Fibrillar Collagens / ultrastructure
  • Image Enhancement / methods*
  • Image Interpretation, Computer-Assisted / methods*
  • In Vitro Techniques
  • Microscopy, Confocal / methods*
  • Microscopy, Polarization / methods*
  • Protein Conformation
  • Rats
  • Rats, Sprague-Dawley
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tendons / metabolism


  • Collagen Type I
  • Fibrillar Collagens